The QIAseq FX DNA Library Kit consists of three, easy-to-follow steps starting from genomic DNA and ending with sequencer-ready NGS libraries. Beginning with combined DNA fragmentation and end modification, sample fragments are prepared allowing for the binding of dual bar-coded adapters. For samples that are less than 100 ng of input DNA or if large amounts of library DNA are required in later applications, an optional DNA amplification step can be performed with reagents conveniently provided in this kit.
DNA fragmentation and adapter ligation
Samples consisting of longer DNA fragments, such as genomic DNA or amplicons from long-range PCR, are first enzymatically sheared into smaller fragments. The median fragment size is dependent on the applications and sequencing read length, and can be adjusted by varying the DNA fragmentation reaction conditions. The fragmented DNA is directly end-repaired, and an 'A’ is added to the 3’ ends during the FX reaction – making the DNA fragments ready for adapter ligation. Following this step, platform-specific adapters are ligated to both ends of the DNA fragments. These adapters contain sequences essential for binding dual bar-coded libraries to a flow cell for sequencing, allowing for PCR amplification of adapter-ligated fragments and for binding of standard Illumina sequencing primers.
To ensure maximum yields from limited amounts of starting material, a high-fidelity amplification step can be performed using the reagents included in the QIAseq FX DNA Library Kit. The proprietary HiFi PCR Master Mix can evenly amplify DNA regions with vastly different GC contents, minimizing sequencing bias caused by PCR.
Dual bar-coded, plate-format adapters are included with the QIAseq FX DNA Library Kit. Each well contains a single-use adapter consisting of a unique combination of two eight-nucleotide identification bar codes. By combining one D5 bar code and one D7 bar code in each ready-to-use adapter, QIAseq FX kits support up to 24-plex or 96-plex pooling prior to sequencing.
Cleanup and removal of adapter-dimers
Following library construction, the reaction cleanup and removal of adapter dimers can be achieved by using Agencourt® AMPure® XP Beads, enabling easy automation on various high throughput automation platforms.